WO2017194903A2 - Particule virale pour le transfert d'arns, notamment dans les cellules impliquées dans la réponse immune - Google Patents
Particule virale pour le transfert d'arns, notamment dans les cellules impliquées dans la réponse immune Download PDFInfo
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Definitions
- the present invention relates to a retroviral system for transferring non-viral RNAs into target cells and more particularly to a retroviral particle capable of delivering multiple RNAs.
- the invention also provides compositions comprising these retroviral particles, kits for their production, related methods of manufacture, and uses of the particles and compositions.
- RNAs into a target cell is a major issue in research and development as in gene therapy.
- virus-derived vectors The use of virus-derived vectors has become a crucial method of gene transfer.
- Viral vectors are now divided into two main categories:
- Integrative vectors which integrate into the recipient genome, and non-integrative vectors, which usually form an extra-chromosomal genetic element.
- Integrative vectors such as gamma-retroviral (RV) and lentiviral (LV) vectors are stably integrated.
- Non-integrative vectors such as adenoviral vectors (ADV) and adeno-associated vectors (AAV) are rapidly cleared from rapidly dividing cells.
- ADV adenoviral vectors
- AAV adeno-associated vectors
- Some factors influencing the choice of a particular vector include its packaging capacity, its range of target or host cells, its gene expression profile, its transduction efficiency and its ability to induce an immune response, which is particularly problematic if repeated transductions are needed.
- RNA Ribonucleic acid
- retroviridae also referred to as the family of retroviruses or RNA viruses.
- a virus of the family Retroviridae has the ability to convert its genome, consisting of RNA, into double-stranded DNA that will be integrated into the genome of the target cell.
- retroviruses are gamma retroviruses and lentiviruses.
- Gag is a gene encoding a polyprotein, the proteins derived from this polyprotein by cleavage being structural proteins involved in the assembly of viruses during replication. These structural proteins are more specifically the matrix protein (MA), the capsid protein (CA) and the nucleocapsid protein (NC).
- MA matrix protein
- CA capsid protein
- NC nucleocapsid protein
- Pol is a gene encoding enzymes integrase, reverse transcriptase and protease.
- MOI multiplicities of infection
- Anti-tumor immunotherapy is a therapeutic strategy that relies on the immune system to eradicate tumor cells.
- New approaches include genetically modifying the patient's cells to induce an immune response and improve targeting.
- RNA transfection and the use of lentiviral vectors emerged respectively as suitable technologies to transfer either a receptor specific for T cells in lymphocytes or an antigen in dendritic cells.
- CPA has a vital role in the immune system and is a key element in an immunotherapy strategy.
- CPA's mission is to present antigenic peptides to cells of the immune system. If the the antigen (s) are immunogenic, the immune system will be specifically activated to eliminate cells or expressing them. This response will therefore be directed against APCs but also against any cell that will express the antigen, which is the case of tumor cells. Any antigen presenting cell (APC) placed in contact with a foreign antigen will process it to associate it with the MHC (Major Histocompatibility Complex) molecules that will allow the presentation of antigenic peptides on its surface.
- APC antigen presenting cell
- a given tumor usually expresses several different antigens, against which it is essential to educate the cells of the immune system in order to make them effective against the tumor. These tumor antigens can also be expressed at different levels depending on the stages of tumor development. Manipulation of the response to several antigens may therefore be crucial in the efficacy of treatment with the modified immune cells.
- the second point is the expression in the target cells of immunomodulatory agents which trigger the maturation or the activation of the modified immune cells.
- These multiple co-expressions within the immune cells will allow to mimic the innate and adaptive immune responses.
- these methods will firstly provide cells expressing specifically antigens and secondly induce their maturation or differentiation which is essential for a complete and effective immune response.
- This means that the most effective therapy will result from the administration of multiple genes to improve therapeutic outcomes, by combining the specificity of the immune response against multiple tumor antigens with stimulating, time-controlled and controlled responses. Delivering at the same time in the target cells or in the body both antigens and immunomodulators and in a single use should provide a clinical benefit.
- the work of the inventors has made it possible to produce a vectorization system capable of delivering several RNAs of interest into the same cell in a single infection.
- the present invention thus relates to a retroviral particle comprising a protein derived from the Gag polyprotein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising a dna RNA sequence.
- each encapsidation sequence being recognized by a binding domain introduced into the protein derived from the Gag polyprotein and / or into the integrase, and at least one of the sequences of interest encapsidated non-viral RNAs include a portion encoding at least one epitope and / or at least one molecular structure specifically recognizing an epitope.
- the retroviral particle according to the invention makes it possible to introduce at least two non-viral RNAs, preferably 3, into a cell by a single infection.
- the introduction of such particles into cells can be performed by an in vivo, in vitro or ex vivo method.
- protein derived from the Gag polyprotein is meant any protein resulting from the cleavage of the Gag polyprotein. More particularly, it is a nucleocapsid protein, a matrix protein (for example, in retroviral particles derived from murine virus of MoMuLV type) or gammaretrovirus-specific p12 protein.
- envelope protein is meant any envelope protein, including pseudotyping envelope protein.
- Ampho envelope protein the ecotropic envelope protein, the Moloney virus envelope protein of murine leukemia (MoMuLV), the envelope protein of the Feline immunodeficiency virus ( FIV), Harvey murine sarcoma virus envelope protein (HaMuSV), murine mammary tumor virus (MuMTV) envelope protein, Rous sarcoma virus (RSV) envelope protein , the measles virus envelope protein (also MV for measles virus), the Gibbon leukemia virus (GalV) envelope protein, the feline endogenous virus protein (RD1 14) or the protein of envelope of vesicular stomatitis virus (VSV-G).
- MoMuLV Moloney virus envelope protein of murine leukemia
- FIV Feline immunodeficiency virus
- HaMuSV Harvey murine sarcoma virus envelope protein
- MuMTV murine mammary tumor virus
- RSV Rous s
- the coat protein is Ampho coat protein, EcoTrope envelope protein, Moloney virus envelope protein of murine leukemia (MoMuLV), Immune deficiency virus envelope protein. Feline (FIV), Harvey murine sarcoma virus envelope protein (HaMuSV), murine mammary tumor virus (MuMTV) envelope protein, Rous sarcoma virus envelope protein ( RSV), the measles virus envelope protein (also MV for measles virus), the virus envelope protein of the Gibbon leukemia (GalV) or vesicular stomatitis virus envelope protein (VSV-G).
- the envelope protein can thus be modified to target certain cell types or certain applications (use of surface receptors as envelope protein).
- envelope protein It is also possible to modify the envelope protein with an antibody, a glycolipid and / or a particular ligand in order to target a particular receptor and / or cell type.
- the envelope protein is the VSV-G protein.
- integrated is meant the enzymatic protein encoded by the pol gene, which allows the integration of the retroviral DNA into the DNA of the retrovirus-infected cell during the replication of said retrovirus.
- encapsidation sequence denotes a motif (sequence and three-dimensional structure) RNA specifically recognized by a binding domain.
- the encapsidation sequence is a rod-loop motif.
- the encapsidation sequence of the retroviral particle is the stem-loop motif of bacteriophage MS2 RNA or PP7 phage such as, for example, that resulting from the sequence ctagaaaacatgaggatcacccatgtctgcag (SEQ ID No. 1) or ctagaaaggagcagacgatatggcgtcgctccctgcag (SEQ ID NO: 2) respectively.
- the stem-loop motif and more particularly the stem-loop motif of the bacteriophage MS2 RNA or that of the PP7 phage RNA may be used alone or repeated several times, preferably from 2 to 25 times, more preferably from 2 to 18 times, for example 6 to 18 times.
- binding domain is meant all or part of a protein specifically binding to the encapsidation sequence linked to the RNA sequence of interest. More particularly, it is a mutant protein or not, defined by a three-dimensional structure, specifically binding to the encapsidation sequence.
- the link domain is a heterologous domain. More preferably, the binding domain is the Coat protein of bacteriophage MS2, PP7 phage or ⁇ -phage, the nun protein of prophage HK022, the protein U1 A or the hPum protein.
- the binding domain is the Coat protein of bacteriophage MS2 or phage PP7.
- the binding domain is the Coat protein of phage PP7
- the sequence thereof is deficient for self-assembly, thanks to a deletion of amino acids 67 to 75 (PCPAFG) (Chao et al. 2008).
- the sequence of the phage PP7 protein PP7 is codon-optimized for human cells, that is to say that the bases of the DNA are chosen to code for amino acids preferentially present in the human species.
- each sequence is meant that the encapsidation sequences may be identical or different, depending on whether these encapsidation sequences are recognized by an identical or different binding domain.
- the retroviral particle according to the invention may comprise one or more binding domains.
- binding domains When several binding domains are introduced, these can be introduced into the Gag polyprotein and / or into the integrase.
- the binding domain not only allows the recognition of the encapsidation sequence but also the encapsidation of the RNAs carrying the encapsidation sequence in the particle (or, in this case, a non-viral RNA linked to a DNA sequence). encapsidation).
- encapsidation is meant the packaging of an RNA in the viral capsid of a viral particle. It will be noted that in the present invention the encapsidation of
- Non-viral RNA is accomplished by recognizing a non-viral packaging signal, particularly other than Psi.
- sequence of interest is meant a sequence coding for or having a function of interest to the user. More particularly, the sequence of interest carried by each of the two encapsidated non-viral RNAs may be identical or different.
- RNAs Since the packaged RNAs are non-viral, these RNAs do not display the recognition sites of the proteins encoded by the pol gene.
- retroviral particles according to the invention comprise a genetic material that is both viral and non-viral:
- the gag gene which can be viral or chimeric. More particularly, the gag gene is chimeric when the binding domain (s) is / are introduced into it.
- the pol gene which may be viral or chimeric. Since the pol gene codes for several enzymes, the sequences relating to these enzymes can be totally or partially deleted, present and non-functional, or present and functional. More particularly, the pol gene is chimeric when the binding domain (s) is / are introduced into the integrase.
- RNAs capable of inducing an effect on gene expression for example by means of shRNA, miRNA, sgRNA, LncRNA or circRNA.
- RNA viruses • Transfer of cellular RNAs, messenger or other RNAs (miRNA 7), genomic replicons of RNA viruses (HCV, etc.) or complete genomes of RNA viruses,
- the viral particles according to the invention make it possible to introduce nucleic acids into target cells capable of inducing:
- one or more epitope (s) and especially one or more antigen (s) in cells involved in the immune response dendritic cells or other antigen presenting cells
- Such activation may be effected by immunomodulatory agents.
- This type of approach can create a favorable microenvironment for triggering an immune response against tumor cells.
- epitope is meant a structure that can be specifically recognized by an antibody or a lymphocyte receptor.
- sequence of interest codes for several epitopes, in particular for an antigen.
- antigen is meant a substance foreign to the body capable of triggering an immune response to eliminate it.
- An antigen is usually a natural or synthetic macromolecule, usually proteins, polysaccharides and their lipid derivatives. It is the recognition of the antigen by the immunocompetent cells, directly or via the antigen presenting cells (APCs), which activates the specific immunity.
- APCs antigen presenting cells
- the same antigen can have several epitopes (identical or different) and thus induce a varied immune response.
- the recognition of the antigen by lymphocytes depends on the nature of the epitope.
- B cells bind directly to conformational epitopes through immunoglobulins in their membrane.
- T cells recognize sequential epitopes, corresponding to an amino acid sequence, presented by the antigen presenting cells.
- the epitope or the antigen comes from a pathogen, in particular a virus, a bacterium, a parasite, a tumor cell, etc.
- the antigen is preferably selected from peptides, proteins, glycoproteins. Indeed, the cellular machinery of the cell (host) can translate the packaged RNA, it can also perform one or more additional post translational modification steps such as glycosylation. Indeed, when the production of the antigen takes place in dendritic cells, the antigen can be matured as the biomarker that made it possible.
- the particle according to the invention allows the introduction of RNA with a size up to 10 kb, it is possible to introduce therein antigenic sequences of consistent size.
- the size of the antigenic sequence is variable. In general, the larger a molecule, the more immunogenic it is.
- the epitope or the antigen is immunogenic, that is to say, it can initiate an immune response.
- immunogen is meant the potential of an antigen to induce an immune response.
- a substance can be antigenic without being immunogenic. This potential depends on the species, the degree of similarity between the antigen and the host molecules, the physicochemical characteristics of the antigen (size, shape, rigidity) and the dose of antigen received.
- the epitope is selected from the list consisting of molecules that are identified as recognized by the variable portion of an antibody or T cell membrane receptor (TCR).
- TCR T cell membrane receptor
- the antigen is a tumor antigen that is to say a molecule specifically present on the surface of the tumor cells, absent or scanty on the surrounding normal cells. More preferably, the antigen is chosen from the group consisting of the antigens of the "cancer testis" group (Ex MAGE-A12, MAGE-A3, MAGE-A10, MAGE-A2, MAGE-A1, CT7 / MAGE-C1, CT10 , SSX4, BRDT, NY-ES01, SSX2, Xagel b, M AGE-A4 Among those tumor tissue outside of ectopic expression by germ cells; differentiation antigens that are expressed in a tissue given by normal cells as well as by the corresponding tumor cells; antigens expressed only in tumor cells that may correspond to mutated antigens (Alpha-actinin-4, NY-FC, P53, elongation factor 2, malic enzyme, EGF-R, Kras, Casp8, ACYN4, ALK-EML14.
- the antigen is chosen from
- melanoma-associated tumor antigens have been classified into three categories depending on whether they are tissue-specific (TRP-1, TRP-1 and TRP-2, gp-100) tissue-specific, for those that are also expressed by a wide variety of cancers (MAGE-1, NY-ESO-1), or they are unique and mutated tumor antigens ( ⁇ -catenin, CDC27).
- a retroviral particle according to the invention comprising an encapsidated non-viral RNA whose sequence of interest comprises a part coding for at least one epitope is particularly advantageous for transducing antigen presenting cells, in particular dendritic cells.
- molecular structure specifically recognizing an epitope or “paratope” is meant a structure capable of establishing a specific interaction with an epitope, including an antigen. It is usually an immunoreceptor consisting of the variable part of an antibody or membrane receptor of lymphocytes. Preferably, it is a TCR (T cell receptor) or CAR (chimeric receptor) type immunoreceptor present on the surface of a T lymphocyte.
- immunoreceptor consisting of the variable part of an antibody or membrane receptor of lymphocytes.
- TCR T cell receptor
- CAR chimeric receptor
- the molecular structure specifically recognizing an epitope is selected from the group consisting of native, modified, or chimeric T cell receptors (TCRs); receptors for B lymphocytes, membrane (BCR) or secreted (immunoglobulins); and receptors of other immune system cells involved in the immune response, such as NK or NKT lymphocytes.
- TCRs native, modified, or chimeric T cell receptors
- BCR membrane
- immunoglobulins immunoglobulins
- NK or NKT lymphocytes receptors of other immune system cells involved in the immune response, such as NK or NKT lymphocytes.
- the retroviral particle according to the invention comprises a nucleocapsid protein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest linked to an encapsidation sequence, each encapsidation sequence being recognized by a binding domain introduced into the core protein and / or integrase.
- RNA of interest without associated integration event in the genome of the target cells.
- RNAs are then released into the cytoplasm and the cellular machinery translates these RNAs directly into protein (s), that is to say without additional steps such as reverse transcription, translocation into the nucleus or integration into the nucleus. genome of the target cell.
- a second binding domain is introduced into the nucleocapsid protein of the retroviral particle according to the invention.
- binding domains can also be introduced into the core protein.
- the nucleocapsid protein is the nucleocapsid protein (NC) of HIV belonging to the chimeric Gag polyprotein, the NC sequence being mutated at the level of the second finger of zinc in order to insert the sequence of the "Coat" protein of the bacteriophage MS2.
- NC nucleocapsid protein
- the coat protein is the VSV-G protein encoding the envelope protein of vesicular stomatitis virus.
- the encapsidation sequence comprises from 2 to 25 repetitions of the stem-loop sequence of MS2, preferentially from 6 to 18 repetitions of the stem-loop sequence, more preferably still from 10 to 14, by example 12 repetitions.
- the stem-loop sequence is as follows: ## STR1 ##
- the nucleocapsid protein is the nucleocapsid protein (NC) of HIV belonging to the chimeric Gag polyprotein, the NC sequence being mutated at the level of the second finger of zinc in order to insert the sequence of the "Coat" protein of the phage PP7.
- NC nucleocapsid protein
- the binding domain is a heterologous domain. More particularly, the binding domain is the Coat protein of bacteriophage MS2, PP7 phage or ⁇ -phage, the nun protein of prophage HK022, the protein U1 A or the hPum protein.
- the at least two encapsidated non-viral RNAs are distinguished by their RNA sequence of interest, that is to say that the RNA sequences of interest included in the two packaged non-viral RNAs are different.
- At least two encapsidated non-viral RNAs carry different encapsidation sequences, each encapsidation sequence respectively corresponding to the first and second binding domain introduced into the integrase.
- At least two of the encapsidated non-viral RNAs have the same encapsidation sequence corresponding to the first binding domain, these two non-viral RNAs possibly being distinguishable by their RNA sequence of interest,
- binding domains can also be introduced into the integrase.
- the nucleocapsid protein is then a chimeric protein derived from a chimeric gag gene.
- the at least two encapsidated non-viral RNAs may carry different encapsidation sequences, each encapsidation sequence corresponding to the binding domains introduced into the integrase and the nucleocapsid protein, respectively.
- At least two of the encapsidated non-viral RNAs have the same encapsidation sequence corresponding to the first binding domain introduced into the integrase, these non-viral RNAs possibly being distinguishable by their RNA sequence of interest,
- the third packaged non-viral RNA carries a different encapsidation sequence, corresponding to a second binding domain introduced into the nucleocapsid protein.
- binding domains can also be introduced into the core protein.
- the invention therefore relates to a retroviral particle comprising a nucleocapsid protein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the packaged non-viral RNAs each comprising an RNA sequence of interest linked to a encapsidation sequence, each sequence encapsidation being recognized by a binding domain introduced into the core protein and / or integrase.
- the at least two non-viral RNAs encapsidated in the retroviral particle are distinguished by their RNA sequence.
- sequence of interest of the other packaged non-viral RNA encodes either for a second epitope or antigen, or for an immunomodulatory protein.
- sequence of interest of the other packaged non-viral RNA encodes a second epitope or antigen
- tumor cells become the target of several independent immune responses.
- dendritic cells capture the antigen from the targeted or infected tissues and then migrate to the nearest secondary lymphoid organ while differentiating.
- the dendritic cells present the antigen to the lymphocytes
- immunomodulatory protein an immunomodulatory agent in the form of a protein.
- This embodiment is particularly advantageous when antigen-presenting cells, and in particular dendritic cells, or lymphocytes are transduced to directly express these immunomodulatory agents and to amplify the immune response.
- the induction of the maturation of antigen-presenting cells, and in particular dendritic cells, concomitant with the expression of an antigen is a particularly promising strategy for inducing an immune response.
- the goal is to prime the immune system to best respond to the target antigen (s).
- Such a priming strategy aimed at pushing the immune system is likely to lead to a strengthening of the immune response.
- the binding domain can be introduced into the integrase, a second binding domain that can be introduced into the nucleocapsid and / or into the integrase.
- the integrase contains nuclear localization sequences (NLS) allowing its localization in the nucleus by the PIC. Therefore, when the encapsidation of the non-viral RNAs is carried out by a binding domain carried by an integrase of a lentivirus, the packaged non-viral RNAs will be transported into the nucleus of the target cell. Indeed, when placing a lentiviral particle in contact with this second embodiment with a target cell, the membrane of the particle and that of the target cell will fuse and allow the release of the capsid content in the cell. target.
- NLS nuclear localization sequences
- integrase which, via the PICs, will allow the import of RNA into the nucleus. This management is particularly interesting for certain applications, such as expression in quiescent cells.
- integrase does not contain these NLS and is therefore localized in the cytoplasm. It is however possible to add in this type of integrase, the NLS sequences in order to induce a nuclear localization of the integrase, and thus of the RNAs supported by this integrase.
- This support is also particularly useful for a CRISPR system, which uses RNA guides that hybridize specifically to the genome of the target cell. Once hybridized, these RNA guides guide a endonuclease (Cas9), which will allow the modification of a specific locus of the genome of the target cell.
- Cas9 endonuclease
- the fact that the transduction is carried out in particular by lentiviral particles, allows a transient expression of the transduced RNAs.
- the particle according to the invention is therefore capable of delivering multiple, optionally genetically distinct, RNAs without an integration event in the genome of the host cells in order to express, on the one hand, multiple different antigenic tumor sequences, for example, and on the other hand immunomodulatory proteins capable of triggering the activation of transduced cells in the immune response mechanism.
- the binding domain is the "Coat" protein of bacteriophage MS2,
- the encapsidation sequence of the non-viral RNAs is a stem-loop sequence of MS2,
- the integrase is a chimeric enzyme protein whose sequence is mutated at the level of the C-terminal domain in order to insert the sequence of the bacteriophage MS2 coat protein.
- the binding domain is the "coat" protein of phage PP7,
- the encapsidation sequence of the non-viral RNAs is a stem-loop sequence of PP7
- integrase is a chimeric enzyme protein whose sequence is mutated at the C-terminal domain in order to insert the sequence of the "coat" protein of PP7 phage.
- the second binding domain introduced into the nucleocapsid may be the phage PP7 coat protein if the first binding domain is the bacteriophage MS2 Coat protein or the second binding domain introduced into the integrase may be the bacteriophage Coat protein. MS2 if the first binding domain is the Coat protein of phage PP7.
- the retroviral particle was therefore modified to contain the "Coat" protein of the bacteriophage MS2 in fusion with the integrase protein (FIG. 1) or the "Coat” protein of the PP7 phage (FIG. XXXVII).
- the encapsidation plasmid p8.74, carrying the pol gene coding for the integrase protein, is modified in order to insert the sequence coding for the Coat protein into the C-terminal of the integrase by assembly PCR.
- the plasmid p8.74 is linearized by PCR then the Coat sequence, previously amplified by PCR, is cloned at the C-terminal level of the integrase, either directly end-to-end or with the addition of a linker.
- the coat protein is the VSV-G protein encoding the envelope protein of vesicular stomatitis virus.
- the encapsidation sequence comprises from 2 to 25 repetitions of the stem-loop sequence of MS2 and / or of PP7, according to the binding domain introduced, preferably from 2 to 18 repeats, more preferably from 2 to 18 repetitions, such as from 6 to 18 repetitions of the stem-loop sequence, more preferably still for the stem-loop sequence of MS2, from 10 to 14, for example 12 repetitions.
- a RLP or MS2RLP or MS2 (NC) -RLP 12X particle is a lentiviral particle formed by encapsidation of RNA bearing the bacteriophage MS2 loop rod motif, repeated 12 times, by the insertion of the bacteriophage MS2 Coat protein into the nucleocapsid,
- a MS2 (NC) -RLP 2X particle is a lentiviral particle formed by encapsidation of RNAs carrying the loop motif of bacteriophage MS2, repeated twice, by the insertion of the bacteriophage MS2 Coat protein into the nucleocapsid,
- an MS2 (IN) -RLP 2X particle is a lentiviral particle formed by encapsidation of RNAs carrying the loop motif of bacteriophage MS2, repeated twice, by the insertion of the bacteriophage MS2 Coat protein into the integrase,
- an MS2 (IN) -RLP 6X particle is a lentiviral particle formed by encapsidation of RNAs carrying the bacteriophage MS2 loop stem motif, repeated 6 times, by the insertion of the bacteriophage MS2 Coat protein into the integrase,
- an MS2 (IN) -RLP 12X particle is a lentiviral particle formed by encapsidation of RNAs carrying the bacteriophage MS2 loop stem motif, repeated 12 times, by the insertion of the bacteriophage MS2 Coat protein into the integrase,
- a PP7 (NC) -RLP 6X particle is a lentiviral particle formed by encapsidation of RNAs carrying the phage stem motif of phage PP7, repeated 6 times, by the insertion of the Coat protein of phage PP7 into the nucleocapsid,
- a particle PP7 (NC) -RLP 12X is a lentiviral particle formed by encapsidation of RNA bearing the phage stem motif of phage PP7, repeated 12 times, by the insertion of the Coat protein of phage PP7 into the nucleocapsid,
- a PP7 (IN) -RLP 6X particle is a lentiviral particle formed by encapsidation of RNAs carrying the phage stem motif of phage PP7, repeated 6 times, by the insertion of the Coat protein of phage PP7 into integrase
- a particle PP7 (IN) -RLP 12X is a lentiviral particle formed by encapsidation of RNAs carrying the phage stem motif of phage PP7, repeated 12 times, by the insertion of the Coat protein of phage PP7 into the integrase
- the particle according to the invention is chosen from MS2 (NC) -RLP 12X, PP7 (NC) -RLP 6X, PP7 (NC) -RLP 2X, MS2 (IN) -RLP 12X, PP7 (IN) - RLP 6X and PP7 (IN) -RLP 2X.
- the invention also relates to compositions comprising particles according to the invention.
- compositions according to the invention are concentrated compositions.
- the compositions are also purified. These compositions may be concentrated and purified according to the process described in Application WO2013 / 014537.
- compositions according to the invention comprise less than 30% of DNA contaminants and less than 45% of protein contaminants relative to the crude supernatant. More particularly, the compositions according to the invention comprise less than 30% of DNA contaminants and less than 2% of protein contaminants relative to the crude supernatant.
- the term "crude supernatant” is intended to mean the supernatant of cell culture (s), comprising retroviral particles according to the invention, after clarification. Such a clarification step is more particularly described hereinafter in the processes for producing the particles according to the invention. When the recovery of the supernatant is carried out in several times, the crude supernatant then corresponds to all the supernatants collected, pooled together and then clarified.
- compositions according to the invention comprise less than 1% of DNA contaminants and less than 1% of protein contaminants relative to the crude supernatant.
- the invention also relates to particle production kits according to the invention and to the methods of manufacturing these kits.
- the invention relates to a kit for producing particles according to the invention, comprising:
- an expression plasmid comprising at least one sequence of interest, for which upstream or downstream of this sequence is inserted an encapsidation sequence
- a packaging plasmid encoding a protein derived from the Gag polyprotein and / or a chimeric integrase, comprising a binding domain making it possible to recognize an encapsidation sequence, and,
- kit may also include instructions for using the plasmids contained in the kit.
- this kit comprises:
- an expression plasmid comprising at least two different non-viral RNA sequences, each RNA sequence comprising a sequence of interest for which upstream or downstream of this sequence of interest is inserted a sequence of encapsidation, or alternatively a first and a second expression plasmid each comprising a sequence of interest upstream or downstream of which is inserted an encapsidation sequence,
- the at least two non-viral RNA sequences are different because the sequences of interest are different and / or the encapsidation sequences are different.
- plasmid is a DNA structure, it is understood that by "expression plasmid comprising at least two non-viral RNA sequences", an expression plasmid encoding at least two non-viral RNA sequences is targeted. .
- the kit expression plasmids contain all the DNA sequences necessary to obtain at least one non-viral RNA by transcription.
- the expression plasmid contains at least one promoter, followed by a DNA sequence of interest (cDNA or DNA which will be transcribed into a non-viral RNA) and an encapsidation sequence (a DNA encoding, for example, MS2 repeat motifs). or PP7) and optionally a stabilizing sequence of the RNA.
- the nucleocapsid protein is the nucleocapsid protein (NC) of HIV belonging to the Gag polyprotein, which NC is mutated at the second finger of zinc to insert the sequence of the protein "Coat" phage PP7.
- NC nucleocapsid protein
- binding domains As examples of different binding domains:
- this kit when the kit aims to produce particles according to the second embodiment, this kit comprises:
- kits according to the invention are also proposed.
- a method of manufacturing a kit according to the invention comprises:
- the at least two non-viral RNA sequences are different because the sequences of interest are different and / or the encapsidation sequences are different.
- the invention also relates to processes for manufacturing the particles according to the invention.
- Such a method comprises a step of co-transfection of cells with:
- these particle manufacturing processes are performed on producer cells grown in serum-free medium and no sodium butyrate induction is performed.
- the supernatant is collected in several times, for example between 3 and 6 times, at specific time intervals, such as at time intervals of the order of the half-life of the retroviral particles.
- the recovery of the supernatant is carried out in 4 to 5 times, at time intervals of the order of 6 to 18 hours, preferably 8 to 16 hours, such as 8h, 12h and / or 16h.
- this collection is performed after changing the culture medium of the cells, this change being preferably carried out at 24 hours post-transfection.
- the particle manufacturing processes of the invention also include a step in which the supernatant is clarified.
- the clarification of the supernatant is carried out by centrifugation.
- the concentration and purification is carried out by frontal ultrafiltration on centrifugation units.
- Tangential ultrafiltration is advantageously carried out on polysulfone hollow fiber cartridges.
- the composition may then undergo an ion exchange chromatography step, in particular anion exchange chromatography.
- the eluate resulting from this chromatography step is then recovered and then concentrated again by frontal ultrafiltration on central centrifugation units.
- a composition resulting from such a process has less than 1% of DNA contaminants and less than 1% of protein contaminants relative to the crude supernatant.
- the co-transfection step can also be carried out with a second encapsidation plasmid coding for:
- a protein derived from the wild-type Gag polyprotein when the first encapsidation plasmid codes for a protein derived from the chimeric Gag polyprotein, and / or a wild-type integrase, when the first encapsidation plasmid codes for a chimeric integrase.
- the ratio of the second encapsidation plasmid to the first encapsidation plasmid is in the range from [10: 90] to [60: 40], preferably in the range of [20: 80] to [50: 80]. 50].
- compositions have less than 30% contaminants
- compositions according to the invention comprise less than 30% of DNA contaminants and less than 2% of protein contaminants relative to the crude supernatant.
- compositions according to the invention comprise less than 1% of DNA contaminants and less than 1% of protein contaminants relative to the crude supernatant.
- the invention relates to the use of a particle according to the invention, or a composition according to the invention for transducing cells, and in particular cells involved in the immune response.
- the use of the particles and compositions according to the invention is particularly advantageous for transducing primary cells and immortalized lines, in order to modify them transiently.
- the cells may be mammalian cells or other eukaryotes.
- the transduction of these cells can be carried out in vivo, in vitro or ex vivo.
- the cells involved in the immune response include antigen presenting cells (APCs) and immune system cells. More particularly, these are monocytes (dendritic cells, macrophages), T or B lymphocytes, natural killers and hematopoietic stem cells.
- APCs antigen presenting cells
- monocytes dendritic cells, macrophages
- T or B lymphocytes T or B lymphocytes
- natural killers hematopoietic stem cells.
- particles or compositions can be described as "vaccines" and are intended to induce cancer or virus-specific effector T cells to reduce the size of tumors or eradicate viral development and also to induce memory T cells that can be avoided. loss of control over the tumor or the virus.
- Introducing multiple and heterogeneous nucleic acids into a target cell is a major issue in research and development as well as in therapy for in vitro, ex vivo and in vivo applications.
- FIG. 1 is a construction diagram of the expression cassette derived from the expression plasmid bearing, as an RNA sequence of interest, a fluorescent reporter, this expression plasmid being used for the production of lentiviral particles MS2RLP according to FIG. invention;
- Figure II shows a construction scheme of the expression cassette derived from the lentiviral encapsidation plasmid p8.74 in which a MS2 Coat binding domain was introduced into the nucleocapsid sequence, this encapsidation plasmid being used for the production of lentiviral particles MS2RLP according to the invention;
- Figure III is a construction scheme of the expression cassette derived from the envelope plasmid
- Figure IV shows three construction schemes of the expression cassette from the integrative lentiviral expression plasmid carrying as a sequence of interest luciferase (IVa), a fluorescent gene (IVb) or Cre (IVc);
- Figure V is a construction scheme of the expression cassette from the p8.74 encapsidation plasmid used for the production of integrative lentiviral (ILV) vectors;
- Figure Via is a diagram illustrating the efficiency of transduction of human lymphocytes with a particle according to the invention.
- Figure VIIa is a diagram illustrating the efficiency of murine lymphocyte transduction with a particle according to the invention.
- Figure VIIIb is a diagram illustrating the efficiency of murine lymphocyte transduction with an integrative lentiviral vector (ILV);
- Figure Villa is a diagram illustrating the efficiency of transduction of immature human dendritic cells with a particle according to the invention.
- the Figure City illustrates the kinetics of expression of ZsGreen in immature human dendritic cells transduced with a particle according to the invention
- Figure VIII illustrates the kinetics of expression of ZsGreen in immature human dendritic cells transduced with an integrative lentiviral vector (ILV);
- the Figure City is a diagram illustrating the phenotyping of immature human dendritic cells transduced with a particle according to the invention.
- Figure VII is a diagram illustrating the phenotyping of immature human dendritic cells transduced with an integrative lentiviral vector (ILV);
- Figure IXa is a diagram illustrating the efficiency of transducing mature human dendritic cells with a particle according to the invention.
- Figure IXb is a diagram illustrating the efficiency of transduction of mature human dendritic cells with an integrative lentiviral vector (ILV);
- Figure IXc is a diagram illustrating the phenotyping of mature human dendritic cells transduced with a particle according to the invention.
- Figure IXd is a diagram illustrating the phenotyping of mature human dendritic cells transduced with an integrative lentiviral vector (ILV);
- Figure Xa is a diagram illustrating the efficiency of mouse bone marrow dendritic cell (BMDC) transduction with a particle according to the invention
- Figure Xb is a diagram illustrating the efficiency of BMDC cell transduction with an integrative lentiviral vector (ILV);
- Figure Xc illustrates the kinetics of expression of ZsGreen in BMDC cells transduced with a particle according to the invention
- Figure Xd illustrates the kinetics of expression of ZsGreen in BMDC cells transduced with an integrative lentiviral vector (ILV);
- Figure Xe is a diagram illustrating the phenotyping of BMDC cells transduced with a particle according to the invention;
- Figure Xf is a diagram illustrating the phenotyping of BMDC cells transduced with an integrative lentiviral vector (ILV);
- FIG. Xla is a construction diagram of the expression cassette derived from the expression plasmid carrying as an RNA sequence of interest, a complete antigenic sequence coding for the MAGE A3 antigen, this expression plasmid being used for the production of lentiviral particles MS2RLP according to the invention;
- FIG. X1b is a construction diagram of the expression cassette derived from the expression plasmid carrying, as an RNA sequence of interest, a partial antigenic sequence coding for the MAGE A3 antigen (PS-MageA3 or PS-MAGEA3) ), this expression plasmid being used for the production of lentiviral particles MS2RLP according to the invention;
- FIG. X1c is a construction diagram of the expression cassette derived from the expression plasmid carrying as an RNA sequence of interest, a partial antigenic sequence coding for the gp100 antigen (PS-gp100 or PS-GP100) , this expression plasmid being used for the production of lentiviral particles MS2RLP according to the invention;
- FIG. Xd1 is a construction diagram of the expression cassette derived from the expression plasmid carrying as an RNA sequence of interest, a partial antigenic sequence coding for tyrosinase (PS-Tyr or PS-TYR), this expression plasmid being used for the production of lentiviral MS2RLP particles according to the invention;
- PS-Tyr partial antigenic sequence coding for tyrosinase
- FIG. XI Ib is an expression plasmid construction diagram carrying as an RNA sequence of interest, a partial antigenic sequence coding for the MAGE A3 antigen (PS-MageA3 or PS-MAGEA3), this plasmid of expression being used for the production of integrative lentiviral vectors (ILV);
- PS-MageA3 or PS-MAGEA3 a partial antigenic sequence coding for the MAGE A3 antigen
- ILV integrative lentiviral vectors
- Figure Xlld is a construction scheme of the expression cassette derived from the expression plasmid carrying as RNA sequence of interest, a partial antigenic sequence coding for tyrosinase (PS-Tyr or PS-TYR), this plasmid of expression being used for the production of integrative lentiviral vectors (ILV);
- PS-Tyr or PS-TYR a partial antigenic sequence coding for tyrosinase
- Figure XIIIa is a diagram illustrating the phenotyping of BMDC cells transduced with a particle according to the invention comprising RNAs encoding MAGE A3 antigen;
- Figure XI Mb is a diagram illustrating the phenotyping of BMDC cells transduced with an integrative lentiviral vector (ILV) comprising an RNA encoding MAGE A3 antigen;
- IMV integrative lentiviral vector
- Figure XIV is a diagram illustrating the monitoring of tumor growth induced by Renca cells expressing MAGE A3 in mice;
- Figure XV shows the pattern of the expression cassette from the expression plasmid carrying as RNA sequence of interest the
- FIG. XVII shows a diagram of the expression cassette derived from the encapsidation plasmid used for the production of lentiviral PP7 (IN) -RLP particles according to the invention, obtained by the modification of the p8.74 lentiviral encapsidation plasmid presented Figure XVI;
- Figure XVIII shows the expression profiles of the murine CD8 marker and CFSE on cells from BABL / c female rats, wild-type or tumor-developed, after magnetic sorting on CD8 expression then CFSE labeling;
- Figure XIX shows the expression profiles of the murine CD8 marker and CFSE obtained with CFSE labeled CD8 + cells grown alone for 5 days (control, bottom), as well as those obtained with CD8 + cells from wild-type (top) or CFSE-labeled (middle) BABL / c mice, then co-cultured for 5 days with untranslated (left) or transduced (right) BMDCs ) with the particles RLP-PS-MAGEA3 / Gp100 / Tyr; the results are expressed as a percentage of proliferated cells;
- Figure XXI is a diagram illustrating the results of an experiment comparing the relative proliferative responses of CD8 + cells co-cultured at the ratio 1 DC: 2T with BMDCs transduced by RLP particles.
- Figure XXII is a diagram illustrating the cellular viability and dose-response of ZsGreen1 expression of BMDC transduced with RLP.ZsGreen1;
- FIG. XXIII is a diagram of the expression cassette derived from an expression plasmid carrying as an RNA sequence of interest a fluorescent reporter, used for the production of lentiviral particles PP7 (NC) -RLP and PP7 (IN). -RLP according to the invention;
- Figure XXIV is a diagram illustrating the cell viability and expression kinetics of BMDC ZsGreen1 transduced with PP7 (IN) -RLP.ZsGreen1 at a dose of 1 ⁇ g p24 / cell;
- Figure XXV is a diagram illustrating the demonstration of MAGEA3-encoding RNA transfer in U937 cells transduced with MAGEA3 RLP, at 5h post-transduction and at 20h post-transduction;
- Figure XXVI is a diagram illustrating the demonstration of the RNA transfer encoding the 3 AATs (PS-MAGEA3, PS-GP100, PS-TYR) in U937 cells transduced with RLP AATs (Antigen Associated with a Tumor or tumor antigen) (PS-MAGEA3, PS-GP100, PS-TYR), at 5h post-transduction;
- Figure XXVII is a diagram illustrating the demonstration of the RNA transfer encoding the 3 AATs (PS-MAGEA3, PS-GP100, PS-TYR), in U937 cells transduced with the RLP AATs (PS-MAGEA3, PS -GP100, PS-TYR), at 20h post-transduction;
- Figure XXVIII is a diagram illustrating the demonstration of the transfer of RNA coding for MAGEA3 in hDC cells transduced with MAGEA3 RLP, at 5 h post-transduction and at 20 h post-transduction
- Figure XXIX is a diagram illustrating the demonstration of the transfer of RNA coding for the 3 AATs (PS-MAGEA3, PS-GP100, PS-TYR), in hDC cells transduced with the RLP AATs (PS-MAGEA3, PS -GP100, PS-TYR), at 5h post-transduction;
- Figure XXX is a diagram illustrating the demonstration of the transfer of RNA coding for the 3 AATs (PS-MAGEA3, PS-GP100, PS-TYR), in hDC cells transduced with the RLP AATs (PS-MAGEA3, PS -GP100, PS-TYR), at 20h post-transduction;
- Figure XXXI shows the scheme of the expression cassette derived from the expression plasmid carrying as RNA sequence of interest the IL12 immunomodulatory protein, formed by the two IL12b (p40) and IL12a (p35) subunits, used for the production of lentiviral particles MS2RLP according to the invention;
- Figure XXXII is a diagram illustrating the demonstration of MAGEA3 and IL12-encoding RNA transfer in U937 cells transduced with RLP-IL-12 / PSMAGEA3 at 5 h post-transduction;
- Figure XXXV is a diagram illustrating the demonstration of the RNA transfer encoding M AGE A3 and IL12 in hDC cells transduced with RLP-IL-12 / PSMAGEA3 at 20 h post-transduction
- Figure XXXVI is a diagram illustrating the quantification of IL12 immunomodulatory protein, secreted by hDCs transduced with RLP-IL12 / MAGEA3 (48 h post-transduction);
- Figure XXXVII shows a construction scheme of the expression cassette from the lentiviral encapsidation plasmid p8.74 in which a PP7 Coat binding domain was introduced into the nucleocapsid sequence, this encapsidation plasmid being used for the production of lentiviral particles PP7RLP according to the invention;
- the multiplicity of infection (MOI) with the RLP particles is expressed in PP or pg of p24 protein per cell and the MOI with the ILV vectors is expressed in TU / ml.
- the transduction tests are carried out for each type of vectors, taking care not to be placed under saturating conditions and to privilege conditions making it possible to visualize a dose effect for each type of particles. It is important to remember that both types of RLP and ILV products are serum-free products and so as to minimize the production of toxic proteins in the supernatant by favoring sequential virion recoveries. Once clarified the supernatants are concentrated and purified by tangential ultrafiltration so as to obtain high purity.
- the sequence of interest may be a DNA encoding a reporter protein such as native Firefly luciferase, a green (ZsGreen1), red (mCherry) or blue (mtBFP) fluorescent protein (whose expression cassette is as described in FIG. ).
- the sequence of interest may be a cDNA encoding a protein, for example an immunogenic protein or an immunomodulatory protein.
- the sequence of interest may also be that of a cDNA, a shRNA, a miRNA, a sgRNA, an LncRNA or a circRNA.
- Envelope plasmid This plasmid carries the gene coding for an envelope protein, which may be, for example, the sequence of the VSV-G gene encoding the envelope protein of vesicular stomatitis virus (the expression cassette is as described in Figure III).
- the expression plasmid carries a promoter-sequence expression cassette of interest.
- the sequence of the gene of interest may be, for example, that of luciferase (FIG. IVa), a fluorescent reporter (FIG. IVb) or CRE recombinase (FIG. IVc).
- This plasmid may contain other elements such as the WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) or the cPPT / CTS sequence. Viral pathogenicity is eliminated by substitution of regions of the viral genome required for retroviral replication by the transgene.
- the respective proportions of the plasmids are as follows: 40% of the expression plasmid, 30% of the plasmid p8.74 (or p8.74AZF), 30% of the plasmid pENV.
- the culture supernatant is replaced by fresh and unsupplemented DMEM medium.
- the cells are incubated at 37 ⁇ C / 5% C02.
- the supernatant is collected four times (32h, 48h, 56h and 72h post transfection). Each recovery is clarified by centrifugation for 5 min at 3000 g before being microfiltered on a 0.45 ⁇ m filter (Stericup, Millipore). All recoveries are then mixed to form the crude supernatant.
- the cells are then trypsinized and the titre (Transduction Unit / mL) is determined by qPCR after extraction of the genomic DNA using the Nucleospin tissue gDNA extraction kit (Macherey-Nagel).
- the titer obtained (TU / mL) by qPCR is normalized by the internal standard whose title was previously determined by FACS.
- the p24 capsid protein is detected directly on the viral supernatant using and following the recommendations of the HIV-1 kit p24 ELISA kit (Perkin Elmer).
- the captured p24 protein is complexed with a biotinylated polyclonal antibody and then detected by streptavidin conjugated with HRP peroxidase.
- the resulting complex is detected spectrophotometrically after incubation with the orthophenylenediamine-HCl substrate (OPD) producing a yellow stain that is directly proportional to the amount of captured p24 protein.
- OPD orthophenylenediamine-HCl substrate
- the absorbance of each well is quantified at the Synergy H1 Hybrid plate reader (Biotek) and calibrated against the absorbance of a standard range of p24 protein.
- the viral titre expressed as physical particles per mL is calculated from the concentration of p24 protein obtained knowing that 1 ⁇ g of p24 protein corresponds to 104 physical particles.
- the cells are resuspended at a concentration of 1.10 6 cells per ml and are plated on a plastic culture support for 2 h at 37 ° C / 5% CO 2.
- Murine lymphocytes are prepared from spleen.
- the spleen of a mouse is recovered after euthanasia of the animal by cervical dislocation.
- the spleen is deposited in a well of a 6-well culture plate and inflated by injection of 1 ml of collagenase solution D (Roche diagnostics) at 2 mg / ml with a syringe mounted with a 26G 1/2 needle.
- the spleen is minced into small pieces in the well with a sterile scalpel in 1 ml of collagenase D solution at 2 mg / ml.
- the 2 ml solution containing the spleen eminents are transferred to a 15 ml tube and incubated.
- the enzymatic digestion is stopped by adding 13 ml of cold MACS buffer (PBS 1X pH 7.4, 0.5% FCS, 2mM EDTA) to a final volume of 15 ml.
- the cell suspension is filtered through a 70 ⁇ cell sieve placed on a 50 ml tube. The volume is completed to 30 ml with cold MACS buffer.
- the cell suspension is centrifuged for 10 minutes at 300 g at + 4 ° C.
- the PBMC are then washed twice with at least 3 volumes of 1 X PBS pH 7.4.
- the cells are centrifuged for 10 minutes at 100 g at room temperature.
- Murine dendritic cells are prepared from bone marrow derived from the lower limbs (BMDC culture).
- BMDC culture bone marrow derived from the lower limbs
- the long bones femurs and shins are recovered from adult mice and transferred to a 50 ml tube containing RPMI 1640 medium supplemented with 1% Penicillin / Streptomycin.
- the bones are emptied of their marrow using a syringe carrying a 26G 1 ⁇ 2 needle and containing RPMI 1640 medium supplemented with 1% Penicillin / Streptomycin.
- the cell suspension is centrifuged for 5 minutes 300 g, + 4 ° C.
- the supernatant is removed and the pellet is taken up in 1 ml of RPMI 1640 medium supplemented with 1% Penicillin / Streptomycin + 3 ml of Gey's solution (155 mM NH4Cl, 1 mM KHC03).
- the suspension is incubated for 5 minutes at room temperature in order to lyse red blood cells contained in the cell suspension. After incubation, the volume is adjusted to 15 ml with RPMI 1640 medium supplemented with 10% Fetal Calf Serum, 1% Penicillin / Streptomycin, 2mM L-Glutamine to stop the reaction and the cell suspension is centrifuged for 5 minutes. g, + 4 ° C.
- the transduction medium is replaced by dendritic cell-specific differentiation medium (SFM, 2mM L-Glutamine, 1% Penicillin / Streptomycin, 200 nM human IL4 and 50 mM human GM-CSF).
- SFM dendritic cell-specific differentiation medium
- This medium allows the culture of immature dendritic cells (iDC).
- the cells are transduced 4 hours post-inoculation by the different vectors (RLP or ILV) to establish a transduction focus on the EF1 .ZsGreen1 cassette.
- the transduced cells are analyzed at different times (1, 2, 3 and 4 days post-transduction).
- the dendritic cells are phenotyped by immunostaining with specific anti-CD1 1 c antibodies, anti-CD209 DC SIGN, anti-CD86 (Miltenyi Biotec) and characterized by flow cytometry.
- the wells are scraped with a pipette cone or scraper, depending on the format of the culture plate, and the cells are transferred to the wells of a conical bottom 96-well culture plate.
- the cells are washed with 1 X PBS pH 7.4, 5% FCS and centrifuged at 300 g for 5 minutes. The supernatant is removed by flipping.
- Viobility TM 485/520 viability marker (Miltenyi Biotec).
- 1 ⁇ of Viobility TM, in solution in DMSO is applied to each well of cells in PBS 10 ⁇ "IX pH 7.4.
- the cells were incubated 20 minutes at room temperature in the dark.
- the cells were centrifuged at 300 g for 5 minutes.
- the supernatant is removed by turning.
- the cells are times in PBS 100 ⁇ "IX pH 7.4, 5% FCS and centrifuged again at 300 g for 5 minutes.
- the supernatant is removed by flipping.
- the cells are taken up in " ⁇ ⁇ PBS " IX pH 7.4, 5% FCS to be labeled with specific antibodies as described below for phenotyping.
- the first dendritic cell (DC) transduction assays were performed on human cells prepared from peripheral blood from healthy human donors.
- Transduction assays were performed on dendritic cells derived from mouse bone marrow (BMDC) as previously described, but including specific staining of dead cells to assess whether toxicity was induced by transduction at increasing doses. ILV or RLP.
- the transduced BMDCs were analyzed at D2 on the percentage of total viable cells as well as on the percentage of cells expressing ZsGreen1 among the cells expressing the dendritic cell-specific CD1 1c marker. The results obtained (FIG.
- XXII show that, 48 hours after the transduction, the specific analysis of the expression of ZsGreen1 in the BMDCs expressing CD1 1 c shows a transduction by the particles RLPs of 50% of the BMDC CD1 1 c + from MOI of 0.5 ⁇ g p24 / cell and 75% by ILV vectors at a MOI of 75. A dose effect is also observed in the analysis of the fluorescence intensity expressed by these cells, with levels of equivalents for the RLPs used at MOI 1 for ILVs or 5 pg p24 / cell for RLPs.
- the expression plasmid carries a promoter-sequence of interest-polyA expression cassette with or without an intron or RNA stabilizing sequence.
- 12 repeats of the stem-loop motif of the MS2 RNA (ctagaaaacatgaggatcacccatgtctgcag, SEQ ID No. 1) were inserted into an expression cassette downstream of the reporter gene.
- the promoter used may be that of CMV or EF1 but other promoters may be used.
- the sequence of interest may encode an antigen or epitope.
- the sequence of interest may be a cDNA encoding a protein, for example an immunogenic protein or an immunomodulatory protein.
- Many biomarkers have been identified on tumor cells and their complete sequence and / or partial sequences are used in immunotherapy.
- the sequence used to express the MAGEA3 antigen may be whole ( Figure Xla) or partial ( Figure Xlb).
- Other partial sequences of tumor biomarkers can be used such as those of gp100 ( Figure Xlc) or tyrosinase ( Figure Xld).
- Many other examples of complete or partial sequences of tumor biomarkers can be used.
- Encapsidation plasmid This plasmid is identical to that described in Example 1 (whose expression cassette is as described in FIG. II).
- Envelope plasmid This plasmid is identical to that described in Example 1 (whose expression cassette is as described in FIG. III).
- Plasmids for the production of lentiviral vectors ILV Plasmids for the production of lentiviral vectors ILV.
- Plasmid expression of a sequence of interest The expression plasmid carries a promoter-sequence expression of interest cassette (whose expression cassette is as described in Figure XII). This plasmid may contain other elements like the WPRE native sequence (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) or the cPPT / CTS sequence. Viral pathogenicity is eliminated by substitution of regions of the viral genome required for retroviral replication by the transgene. As before, the sequence of interest can encode an entire antigenic protein, an antigen or an epitope. Many biomarkers have been identified on tumor cells and their complete sequence and / or partial sequences are used in immunotherapy.
- sequences used to express MAGEA3 can be integer ( Figure Xlla) or partial ( Figure Xllb).
- Other partial sequences of tumor biomarkers can be used such as those of gp100 ( Figure Xllc) or tyrosinase ( Figure Xlld).
- Many other examples of complete or partial sequences of tumor biomarkers can be used.
- Encapsidation plasmid This plasmid is identical to that described in Example 1 (whose expression cassette is as described in FIG. V).
- Plasmid of the envelope (pENV): This plasmid is identical to that described in Example 1 (whose expression cassette is as described in Figure III).
- the batch titration is carried out according to a method identical to that described in Example 1.
- BMDC Murine Dendritic Cells
- the preparation of the murine dendritic cells is carried out according to a method identical to that described in Example 1. 1. Transduction of murine dendritic cells with a single tumor antigen by A3-IRES-GFP RLP-MAGE
- the dendritic cells transduced by the A3-IRES-GFP RLP.MAGE particle have been focused on the characterization of the fluorescence expression analyzed by flow cytometry according to a growing range of RLP particles ranging from from 0.1 ⁇ g p24 to 5 ⁇ g p24 and at different assay times (1, 2, 3 and 4 days post-transduction).
- a negative transduction control is prepared with the medium containing only the Polybrene® transducer at 4 ⁇ g mL (Sigma).
- the dendritic cells are phenotyped by immunostaining with specific antibodies anti-CD1 1 c, anti-CD86, anti-CD80 (Miltenyi Biotec) and characterized by flow cytometry.
- the dendritic cells transduced by the ILV-MAGE A3-IRES-GFP vector were the subject of a focus related to the characterization of the fluorescence expression analyzed by flow cytometry according to a growing range of ILV lentiviral particles. ranging from a multiplicity of MOI infection of 5 to 75 and at different analysis times (1, 2, 3 and 4 days post-transduction).
- a negative transduction control is prepared with the medium containing only the Polybrene® transducer at 4 ⁇ g / mL (Sigma).
- the dendritic cells are phenotyped by immunostaining with specific antibodies anti-CD1 1 c, anti-CD86, anti-CD80 (Miltenyi Biotec) and characterized by flow cytometry.
- the cells are transferred to the wells of a conical bottom 96-well culture plate.
- the cells are washed with 1 X PBS pH 7.4, 5% FCS and centrifuged at 300 xg for 5 minutes. The supernatant is removed by flipping.
- the dendritic cells are phenotyped by immunostaining with the following specific antibodies conjugated to fluorochromes: anti-CD1 1 c, anti-CD86, anti-CD80 (Miltenyi Biotec). For this, mixtures of antibody solution are made and 50 ⁇ per well are deposited on the dendritic cells recovered. The cells are incubated for 20 minutes at room temperature in the dark. 50 ⁇ PBS "IX pH 7.4, 5% FCS are added and the cells are centrifuged at 300 g for 5 minutes. The supernatant is removed by turning.
- the cells were resuspended in" PBS ⁇ ⁇ "IX pH 7.4
- the cells are centrifuged at 300 g for 5 minutes, the supernatant is removed by inversion
- the cells are taken up in " ⁇ ⁇ of PBS " IX pH 7.4, 5% FCS and analyzed by flow cytometry.
- Immunolabeled dendritic cells are analyzed by flow cytometry (Miltenyi Biotec) and analyzed. The samples were sized according to their size (SSC) and granulosity (FSC). The cells are characterized on the MacsQuantVYB (Miltenyi Biotec) and analyzed with the MacsQuant software (Miltenyi Biotec).
- This model uses the Renca type murine kidney cancer (Balb / c) tumor cells. These murine cells are previously transduced with the vector ILV.EF1 .MAGE A3.IRES.GFP (MOI 100) so as to express the MAGE A3 antigen which is of human origin.
- the model then consists of reimplanting a predefined number of these cells (1 .10 6 cells) subcutaneously into the flank of adult Balb / c syngeneic mice.
- a preliminary study determined the number of cells to be re-implanted to generate a tumor that develops in all animals, and without reaching endpoints (tumor> 2mm3, weight loss of animals> 20% of weight on day 1). implantation of the tumor) too quickly.
- the animals come from an approved breeder (January Europe or Charles River Labs) and the protocol put in place for all these experiments has been submitted and approved by a local ethics committee (EAEC-122 ethics committee) .
- the BMDCs are transduced as described in Example 1 (2.4) with the particles of the RLP type (Example 1, section 2.4.1) or ILV (Example 1, section 2.4.2) and are reimplanted the day after the transduction in the Balb / c mouse, by intradermal approach at the level of the inguinal ganglion situated on the same side as that of tumor reimplantation. Different amounts of cells can be reimplanted to evaluate their therapeutic potency in this model. 3. Monitoring tumor development
- BMDC murine dendritic cells
- BMDCs were transduced by RLP.MAGE A3.IRES.GFP particles or an ILV.EF1 .MAGE A3.IRES.GFP vector under the optimal conditions determined by the ZsGreenl fluorescence assays.
- a range of MOIs were made in both cases and the cells were phenotyped on the third day for the expression of markers specific for dendritic cells ( Figure XI Ia and XIIIb). As before, these analyzes show that the phenotype is not altered by transduction, regardless of the MOI applied.
- BMDCs transduced by RLP.ZsGreen1 or RLP.MAGE A3 particles were also used to test the development of tumors expressing MAGE A3 in vivo, in the Balb / c mouse.
- Two groups of mice received Renca syngeneic tumor cells (0.75 ⁇ 10 6 cells), themselves previously modified to express the MAGE A3 tumor antigen of human origin. The same day these animals were received intradermally, near the corresponding inguinal ganglion, a suspension of BMDC (1 x 10e5 cells) modified by RLP-ZsGreenl for the control group or by RLP.MAGE A3 for the test group.
- the follow-up of the tumor development then showed that the animals that received the BMDC.MAGE A3 did not develop tumors, unlike animals in the control group ( Figure XIV).
- the expression plasmid carries a promoter-sequence of interest-polyA expression cassette (FIG. XV or XXIII) with or without an intronic or stabilizing sequence.
- RNA In order to transport the mRNAs in the lentiviral particles, 2 repetitions of the stem-loop motif of the PP7 RNA (ctagaaaggagcagacgatatggcgtcgctccctgcag SEQ ID No. 2 and ctagaaaccagcagagcatatgggctcgctggctgcag SEQ ID No. 3) were inserted into an expression cassette. downstream of the reporter gene ( Figure XV or XXIII).
- the promoter used may be that of CMV or EF1 ( Figure XV or XXIII) but other promoters may be used.
- the sequence of interest may be a DNA encoding a reporter protein such as native Firefly Luciferase (Figure XV), a green fluorescent protein (ZsGreen1) ( Figure XXIII), red (mCherry) or blue (mtBFP), or a cDNA coding a protein, for example CRE protein.
- the cDNA encodes an immunogenic protein or an immunomodulatory protein.
- the sequence of interest may also be that of a shRNA, a miRNA, a sgRNA, an LncRNA or a circRNA.
- Packaging plasmid The lentiviral particle was modified to contain within the integrase, the sequence of the protein "Coat" bacteriophage PP7.
- the encapsidation plasmid p8.74, carrying the genes encoding the structure and function proteins (Gag, Pol), used for the production of the PP7 (IN) -RLP 2X particles is modified according to the strategy illustrated in FIG. XVI: this plasmid p8.74 is used to generate, by assembly PCR, a plasmid on which the Coat protein of phage PP7 is fused to the terminal C domain of the integrase.
- This fusion obtained by Hpal cloning, makes it possible to generate the plasmid P8.74-POL-PP7 Coat.
- the construction whose expression cassette is illustrated in FIG. XVII is thus obtained.
- the coding sequence Pol can be deleted or mutated in certain functional elements, for example the sequence encoding the reverse transcriptase (RT).
- the lentiviral particles are produced as described in Example 1, concentrated and purified according to the method P2. The particles are titrated as described in Example 1.
- BMDC Murine Dendritic Cells
- the preparation of the murine dendritic cells is carried out according to a method identical to that described in Example 1.
- the dendritic cells are transduced by the PP7 (IN) -RLP.ZsGreen1 vector as described in Example 1 (section 2.4), to a MOI of 1 ⁇ g p24 per cell and analyzed for their viability and the expression of ZsGreen1 fluorescence. by flow cytometry at different analysis times (1, 2 and 3 days post-transduction).
- a negative transduction control is prepared with only the medium containing the Polybrene® transducer at 4 ⁇ g / mL (Sigma).
- the viability of dendritic cells is achieved by specific immunolabeling "Viobility TM 485/520" (Miltenyi Biotec) and analyzed by flow cytometry.
- the viability of the dendritic cells is carried out by specific immunostaining with "Viobility TM 485/520" (Miltenyi Biotec) according to an identical procedure to that described in Example 1.
- Immunolabeled dendritic cells are analyzed by flow cytometry (Miltenyi Biotec) and analyzed. The samples were sized according to their size (SSC) and granulosity (FSC). Viobility TM labeled cells are dead, so viable cells are negative cells (exclusion labeling). The cells are characterized on the MacsQuantVYB (Miltenyi Biotec) and analyzed with the MacsQuant software (Miltenyi Biotec).
- Figure XXIV shows the measurement of cell viability and expression kinetics of BMDC ZsGreen1 (CPA) transduced with PP7 (IN) - RLP.ZsGreen1 particles at a dose of 1 ⁇ g p24 per cell.
- the proportion of murine BMDCs transduced with PP7 (IN) -RLP particles that express ZsGreen1 is 70%, from 24h post-transduction and stably up to 3 days. The cells remain at the same level of viability over the 3 days of analysis, only the intensity of expression decreases over time. It is therefore possible to carry RNA in lentiviral particles with PP7-Coat in integrase.
- the PP7 (IN) -RLP particles are used to convey RNAs carrying antigenic sequences in APCs, such as BMDCs.
- the expression plasmids carry a promoter-sequence of interest-polyA expression cassette with or without an intron or RNA stabilizing sequence.
- 12 repeats of the stem-loop motif of the MS2 RNA (ctagaaaacatgaggatcacccatgtctgcag, SEQ ID No. 1) were inserted into an expression cassette downstream of the reporter gene.
- the promoter used may be that of CMV or EF1 but other promoters may be used.
- the sequence of interest can encode an antigen or epitope. Many biomarkers have been identified on tumor cells and their complete sequence and / or partial sequences are used in immunotherapy.
- the sequence used to express the MAGEA3 antigen may be whole ( Figure Xla) or partial ( Figure Xlb).
- Other partial sequences of biomarkers can be used such as those of gp100 ( Figure Xlc) or tyrosinase ( Figure Xld).
- Many other examples of complete or partial sequences of tumor biomarkers can be used.
- Encapsidation plasmid This plasmid is identical to that described in Example 1 (FIG. II).
- Plasmid of the envelope (pENV): This plasmid is identical to that described in Example 1 ( Figure III).
- Batch production and titration is performed according to method P2 described in Example 1, using one or more of the following expression plasmids:
- the respective proportions of the plasmids are as follows: 40% of the expression plasmid or expression plasmids (in the case of RLP-AATs), 30% of the plasmid p8.74 (or p8.74AZF), 30% of the plasmid pENV.
- the cells are resuspended and taken in 15 ml tube, then centrifuged at 200 g for 5 minutes to pellet. The supernatant is discarded and the cells are washed in 1 ml of 1 X PBS before being centrifuged again at 200 g for 5 minutes. The supernatant is discarded and the cell pellet is resuspended in 1 ml of 0.05% Trypsin-0.53mM EDTA (Corning). The cells are incubated for 5 minutes at 37 ° C. At the end of the incubation, 2 ml of complete RPMI culture medium is added to the cell suspension.
- Sense oligonucleotides are:
- the transduction of hDC is carried out according to a method identical to that described in Example 1 (part II, section 2.3).
- the cells are transduced with 4 ⁇ g of p24 / cell and are analyzed at different times (5h, 20h post-transduction).
- the expression plasmids carry a promoter-sequence of interest-polyA expression cassette with or without an intron or RNA stabilizing sequence, as described. in Figures Xllb, XI and Xlld.
- 12 repeats of the stem-loop motif of the MS2 RNA (ctagaaaacatgaggatcacccatgtctgcag, SEQ ID No. 1) were inserted into an expression cassette downstream of the reporter gene.
- the promoter used may be that of CMV or EF1 but other promoters may be used.
- the sequence of interest may encode an antigen or epitope.
- Many biomarkers have been identified on tumor cells and their complete sequence and / or partial sequences are used in immunotherapy. Many other examples of complete or partial sequences of tumor biomarkers can be used.
- Envelope plasmid This plasmid carries the gene coding for an envelope protein, which may be, for example, the sequence of the VSV-G gene encoding the envelope protein of vesicular stomatitis virus (the expression cassette is as described in Figure III).
- This model is that described in Example 2, for which the Renca tumor cells that have been previously transduced to MOI 100 are used with the ILV.EF1 .PS-MAGEA3 / Gp100 / Tyr vector containing a mixture of the 3 expression plasmids. whose expression cassettes are as described in Figures Xllb, c and d so as to stably express the antigenic peptides MAGE A3, gp100 and tyrosinase, all three of human origin.
- the model then consists of reimplanting 0.75 ⁇ 10 6 of these cells subcutaneously into the flank of BALB / c adult syngeneic background mice. The animals come from an approved breeder (January Europe) and the protocol put in place for all these experiments was submitted and approved by a local ethics committee (EAEC-122 ethics committee).
- the murine lymphocytes are prepared from spleen of wild-type BALB / c mice or from mice having developed a Renca tumor> 1 mm 3 .
- the spleen is recovered after euthanasia of the animal by cervical dislocation. It is dissociated mechanically by crushing on a 70 ⁇ cell sieve.
- the cell suspension is centrifuged for 5 minutes at 300 g at + 4 ° C.
- the cell pellet is then taken up in 10 ml of cold MACS buffer, the suspension filtered on a 40 ⁇ cell sieve placed on a 50 ml tube and then a cell count is performed.
- the reaction is stopped by diluting the cells 5 times in RPMI supplemented with 1% Fetal Calf Serum and incubating the suspension again for 5 minutes.
- the cell suspension is then centrifuged for 5 minutes at 300 g at room temperature and the cells resuspended at a concentration of 1.6 ⁇ 10 6 cells per ml in RPMI 1640 medium supplemented with 10% of Fetal Calf Serum, 1% Penicillin / Streptomycin, 2mM L-Glutamine to be co-cultured at different ratios with murine BMDCs, in a ULA (Corning) flat-bottomed 96-well plate.
- ULA Corning
- CFSE-labeled CD8 + T lymphocytes from BALB / c mice that were wild-type or developed a Renca tumor, are cultured in the presence of syngenic murine BMDCs transduced either with the RLP.MAGE A3.IRES.GFP particles or with the RLP-PS particles. -MAGEA3 / Gp100 / Tyr, or with non-transduced BMDCs. Two ratios were tested for co-cultures with BMDCs transduced with RLP-PS-MAGEA3./Gp100/Tyr: 1 BMDC particles for 2 CD8 + T cells (1 DC: 2T) or 1 BMDC for 4 T cells CD8 + (1 DC: 4T).
- the cells were resuspended in PBS 100 ⁇ "IX pH 7.4, 5% FCS and analyzed by flow cytometry, both according to the expression CD8 marker (which allows to discriminate BMDC still present in suspensions) and the expression of CFSE, whose relative fluorescence level is halved at each cell division, which allows to quantify the proliferative response of cells .
- Figure XIX shows the expression profiles of CFSE obtained with CD8 + T cells grown alone as well as those obtained after 5 days of co-culture with non-transduced or transduced BMDCs with particles RLP-PS-MAGEA3 / Gp100 / Tyr .
- Immunolabeled lymphocyte cells are analyzed by flow cytometry (Miltenyi Biotec). The samples were sized according to their size (SSC) and granulosity (FSC). The cells are characterized on the MacsQuantVYB (Miltenyi Biotec) and analyzed with the MacsQuant software (Miltenyi Biotec).
- Figure XIX shows that CFSE-tagged CD8 + T cells co-cultured for 5 days with BMDC (DC: 2T ratio) respond by proliferating. This translates into cytometry by a decrease in the fluorescence intensity of CFSE labeling to the left, on one or more peaks. The analysis relates to the percentage of cells in these peaks. The profiles shown are representative of 3 experiments.
- the response of CD8 + T cells from a wild-type mouse is similar whether they are in contact with transduced or non-transduced BMDCs. This phenomenon is explained by the fact that na ⁇ ve lymphocytes placed in contact with dendritic cells are able to proliferate, even in the absence of antigen (Q.
- the relative proliferative response is given by the ratio of the percentage of proliferating cells in response to BMDCs transduced with RLP-PS-MAGEA3 / Gp100 / Tyr particles relative to the percentage of cells responding non-specifically to non-transduced BMDCs.
- CD8 + T cells from mice that have developed a Renca tumor expressing the 3 human tumor-associated antigens (AATs) respond specifically to further stimulation by these AATs (2.67 for 1 DC: 2T co-cultures and 3.90 for co-cultures 1 DC: 4T) whereas CD8 + T lymphocytes from naive mice respond similarly to BMDCs transduced with RLP-PS-MAGEA3 / Gp100 / Tyr particles (1.59 for co-cultures 1 DC: 2T and 1.78 for co-cultures 1 DC: 4T).
- Figure XXI compares the relative proliferative responses of CD8 + T cells co-cultured in condition 1 DC: 2T with BMDCs transduced by RLP.MAGEA3.IRES.GFP particles or transduced by particles RLP-PS-MAGEA3 / gp100 / Tire.
- FIG. XX these analyzes show that CD8 + T lymphocytes derived from mice having developed a Renca tumor carrying the 3 AATs respond more than the CD8 + T lymphocytes from wild mice to the BMDCs transduced with the particles RLP-PS. -MAGEA3 / Gp100 / Tire.
- BMDCs transduced with the particles RLP-PS-MAGEA3 / Gp100 / Tyr are therefore well capable of presenting the antigenic peptides corresponding to the 3 AATs (MAGE A3, gp100 and tyrosinase).
- the RNA molecules corresponding to the 3 peptides are therefore supported by the cell machinery specific to the antigen-presenting cells which present these peptides on the surface sufficiently long and efficiently so that the CD8 + T lymphocytes which meet them can be activated.
- the expression plasmid carries a promoter-sequence of interest-polyA expression cassette with or without an intron or RNA stabilizing sequence.
- 12 repeats of the stem-loop motif of the MS2 RNA (ctagaaaacatgaggatcacccatgtctgcag, SEQ ID No. 1) were inserted into an expression cassette downstream of the reporter gene.
- the promoter used may be that of CMV or EF1 but other promoters may be used.
- the sequence of interest may encode an antigen or epitope. Many biomarkers have been identified on tumor cells and their complete sequence and / or partial sequences are used in immunotherapy.
- the sequence used to express the MAGEA3 antigen may be whole ( Figure Xla) or partial ( Figure Xlb).
- the sequence of interest may also encode an immunomodulatory protein, such as a cytokine or an interleukin such as IL-12 for example.
- Encapsidation plasmid This plasmid is identical to that described in Example 1 (whose expression cassette is as described in FIG. II).
- Plasmid of the envelope (pENV): This plasmid is identical to that described in Example 1 (whose expression cassette is as described in Figure III).
- the respective proportions of the plasmids are as follows: 40% of the expression plasmids, 30% of the plasmid p8.74 (or p8.74AZF), 30% of the plasmid pENV. II. Transduction of cells of the immune system
- the U937 cell line was cultured under the conditions described in Example 4 (II, paragraph 1).
- Example 4 After 5 hours and 20 hours post-transduction, the cells are recovered and treated according to the method described in Example 4 (II, paragraph 1 .2).
- Each dry pellet is treated according to the method described in Example 4 (II.1.1) to extract and purify the total RNA.
- RNAs into complementary DNA are carried out according to the method described in Example 4 (II, paragraph 1 .4). 1 .5. Amplification of cDNAs by RT-oPCR
- the amplifications are carried out on 5 ⁇ l of cDNA diluted 1/10 in ultrapure water, with the real-time PCR mix, SYBR® Premix Ex Taq (Takara-cat RR420L), in the presence of 200nM sense oligonucleotide. and antisense in a final volume of 20 ⁇ . To each well is added 5 ⁇ l of cDNA diluted 1/1 Oth.
- the real-time PCR is carried out with a StepOnePlus thermal cycler (AppliedBiosystems) with SYBR ⁇ Green chemistry according to the following protocol: 1 cycle of 30 seconds at 95.0 ° C and then 40 cycles comprising 5 seconds at 95.0 ⁇ and 30 seconds at 60.0 ⁇ C.
- Sense oligonucleotides are:
- Q-hGAPDH-S GACAAGCTTCCCGTTCTCAG
- Q-PSMAGEDC-F TAGCACTCTTTGAGGATCTAATGA
- the AntiSense oligonucleotides used are:
- the associations of the pairs of oligonucleotides to be used are the following according to the targets:
- the preparation of the immature human dendritic cells is carried out according to a method identical to that described in Example 1 (Part II, Section 2.1).
- the transduction of human dendritic cells is carried out according to a method identical to that described in Example 1 (part II, section 2.3).
- the cells are transduced with 4 ⁇ g of p24 / cell and are analyzed at different times (5h, 20h post-transduction).
- a negative transduction control is prepared with the medium containing only the Polybrene® transducing agent at 4 ⁇ g -1 (Sigma).
- the dendritic cells are analyzed on the day of transduction and at different analysis times, by RT-qPCR on the RNAs derived from transduced cell pellets.
- RNA expression For the analysis of RNA expression, the recovery of human dendritic cells at different times after transduction is carried out according to the protocol described in Example 4 (II, paragraph 2.2). The cells are frozen at -10 ° in the form of dry pellets. For the analysis of IL12 secretion, culture supernatants cells are taken at 20h and 48h post-transduction times.
- the supernatants are stored at -20 ° C.
- RNAs The preparation of the RNAs is carried out according to a method identical to that described in Example 4 (II, paragraph 2.3).
- Retro-transcription of the RNAs into complementary DNA is carried out on 500ng of RNA according to the method described in Example 4 (II, paragraph 2.4).
- the ELISA test for the quantification of the secretion of IL12 in the cell culture supernatants is carried out according to the protocol provided with the kit "Human IL-12 Standard ABTS ELISA Development Kit” (Peprotech).
- RT-qPCR The specific RT-qPCR analysis makes it possible to demonstrate the transfer of RNA coding for an antigen and an immunomodulatory protein, by RLP particles in APCs, in particular in U937 cells and in immature human dendritic cells (hDC ), at 5h and 20h post-transduction (respectively Figures XXXII and XXXIII, XXXIV and XXXV).
- the results obtained are analyzed and interpreted according to the presence or not of a specific and significant amplification during real-time quantitative PCR (RT-qPCR).
- the results are normalized with respect to the values obtained for non-transduced control cells (U937 NT and hDC NT). These controls express endogenous RNA encoding NL12 (results not shown). It is known that dendritic cells produce IL12, which plays a role in T cell response in vivo.
- the RLP particles are therefore effective for the transfer into APCs (lines and primary cells) of different RNA molecules encoding an antigenic peptide and for an immunomodulatory protein such as IL-12.
- the RLP particles are therefore part of an immunotherapy strategy, based on a modulation of the specificity of the immune response, by the transfer into APCs of RNAs encoding antigens and for immunomodulatory proteins.
- the expression plasmid carries an expression cassette as described in FIG. XXIII with or without an intron or RNA stabilizing sequence.
- 2 repetitions of the stem-loop motif of the PP7 RNA sequence ctagaaaggagcagacgatatggcgtcgctccctgcag (SEQ ID No. 2) followed by the sequence ctagaaaccagcagagcatatgggctcgctggctgcag (SEQ ID No. 3) were inserted within an expression cassette downstream of the reporter gene ( Figure XXIII).
- the promoter used may be that of CMV or EF1 ( Figure XXIII).
- the sequence of interest may be a DNA encoding a reporter protein such as Luciferase Firefly native ( Figure XV), a green fluorescent protein (ZsGreenl), red (mCherry) or blue (mtBFP), or a cDNA encoding a protein, for example CRE protein.
- the cDNA encodes an immunogenic protein or an immunomodulatory protein.
- the sequence of interest may also be that of a shRNA, a miRNA, a sgRNA, an LncRNA or a circRNA.
- the lentiviral particle was modified to contain, within the nucleocapsid protein, instead of the second Zn finger domain, the sequence of the "Coat" protein of the bacteriophage PP7 (FIG. XXXVII).
- the encapsidation plasmid p8.74, carrying the genes encoding the structure and function proteins (Gag, Pol), used for the production of the PP7RLP particles is modified according to the following strategy: this plasmid p8.74 is used to generate, by assembly PCR, a plasmid lacking the second zinc finger of the p8.74AZF nucleocapsid protein.
- the second zinc finger is substituted by the Coat protein of phage MS2 by Hpal cloning, to generate the plasmid p8.74AZF-PP7-Coat.
- the Pol coding sequence can be deleted or mutated in certain functional elements, for example the reverse transcriptase (RT) or integrase (IN) coding sequence, without altering the function of PP7RLPs.
- Envelope plasmid This plasmid carries the gene coding for an envelope protein, which may be VSV-G encoding the vesicular stomatitis virus envelope protein (whose expression cassette is as depicted in Figure III).
- the lentiviral particles are produced as described in Example 1, according to the P2 method, using the expression plasmid pcDNA-EF1. Fluorescent.PP7 2X reporter depicted in Figure XXIII.
- BMDC Murine Dendritic Cells
- the preparation of the murine dendritic cells is carried out according to a method identical to that described in Example 1.
- the dendritic cells are transduced by the vector PP7 (NC) -RLP.ZsGreen1 as described in Example 1 (II, section 2.4), at a dose of 1 ⁇ g p24 per cell and analyzed for their viability and the expression of the ZsGreenI fluorescence by flow cytometry at different analysis times (1, 2 and 3 days post-transduction).
- a negative transduction control is prepared with only the medium containing the Polybrene® transducer at 4 ⁇ g -1 (Sigma).
- the cells are transferred to the wells of a conical bottom 96-well culture plate.
- the cells were washed with PBS solution "IX pH 7.4 and centrifuged at 300 g for 5 minutes. The supernatant is removed by turning.
- the viability of the dendritic cells is carried out by specific immunolabeling with "Viobility TM 485/520" (Miltenyi Biotec) according to a method identical to that described in Example 1 in Section II.3.3. 2.2 Flow cytometry
- Immunolabeled dendritic cells are analyzed by flow cytometry (Miltenyi Biotec) and analyzed. The samples were sized according to their size (SSC) and granulosity (FSC). Viobility TM labeled cells are dead, so viable cells are negative cells (exclusion labeling). The cells are characterized on the MacsQuantVYB (Miltenyi Biotec) and analyzed with the MacsQuant software (Miltenyi Biotec).
- Plasmids for the production of a lentiviral particle MS2RLP according to the invention are identical to Plasmids for the production of a lentiviral particle MS2RLP according to the invention.
- MS2RLP particles The production of MS2RLP particles is carried out by transfection of the following four plasmids:
- p8.74AZF-MS2-Coat (whose expression cassette is illustrated in FIG. II) and the plasmid p8.74 (whose expression cassette is illustrated in FIG. V), using four different ratios of the two plasmids of FIG. encapsidation, respectively [100% - 0%]; [90% - 10%] [80% - 20%] and [50% - 50%];
- the lentiviral particles and the lentiviral vectors are concentrated and purified according to the method P1 described in Example 1. 3. Titration
- Lentiviral particles and lentiviral vectors are titrated as described in Example 1.
- the culture supernatants are recovered and then concentrated according to the method P1, as described in Example 1, and titrated by quantification of the p24 protein. as described in Example 1.
- the equivalent of 15ng of p24 are loaded for each ratio condition of plasmids p8.74AZF-MS2- Coat / p8.74 on a 4/12% SDS-PAGE denaturing gel and then migrated for 1 hour at 200V in MOPS1 X buffer.
- the proteins After transfer onto a nylon membrane, the proteins are hybridized with an anti-p24 antibody (clone 39 / 5.4A , Abcam).
- the western blot is revealed using the PierceTM Fast Western Blot Kit, ECL Substrate (Pierce). The visualization of the bands is performed by chemiluminescence on an autoradiography film.
- This example aims to show that it is possible to improve the functionality of the MS2RLP and PP7RLP particles for the transfer of RNA coding for different antigens or for the transfer of RNA coding for antigens and for an immunomodulatory protein.
- the plasmid p8.74AZF-MS2 allows the expression of a GAG precursor comprising the bacteriophage Coat protein in place of the second Zinc finger of the Nucleocapsid protein. This Coat protein is likely to disturb the processing of the GAG precursor, when it is cleaved into three proteins: Matrix Protein, Capside Protein and Nucleocapsid Protein. These three proteins are essential for the structure of viral particles.
- the addition of the wild-type GAG precursor by the plasmid p8.74 in addition to the p8.74AZF-MS2-Coat encapsidation plasmid during the production of the MS2RLP-ZsGreen1 12X particles could make it possible to increase the maturation of the GAG precursor when it is expressed by the plasmid p8.74AZF-MS2, and thus, increase the functionality of the particles MS2RLP and PP7RLP.
- the objective of this experiment is to evaluate the impact of the plasmid p8.74 when it is co-transfected, in the production cells of the particles MS2RLP and PP7RLP, at the same time as the p8.74AZF-encapsidation plasmid.
- MS2-Coat to improve the processing of the GAG precursor, and thus make the final particles more functional for target cell transduction.
- Coat / p8.74 are tested: 100/0; 90/10; 80/20 and 50/50.
- An integrative vector ILV expressing ZsGreen1 is used as a control.
- the cells are transduced in two amounts of p24 / ml: 2 ⁇ g p24 / cell ( Figure XXXIX) and 10 ⁇ g p24 / cell ( Figure XXXX).
- the cells transduced by the MS2RLP-ZsGreen1 12X particles produced with 50% of the encapsidation plasmid p8.74AZF-MS2-Coat and 50% of the plasmid p8.74 have a fluorescence intensity of 7.76 whereas that of the cells transduced by the MS2RLP-ZsGreen1 12X particles produced solely with the encapsidation plasmid p8.74AZF-MS2-Coat is 3.5.
- Figure XXXX shows the same result in terms of percentage of transduced cells.
- the cells transduced by the MS2RLP-ZsGreen1 12X particles produced with 50% of the p8.74AZF-MS2-Coat encapsidation plasmid and 50% of the plasmid p8.74 have a fluorescence intensity of 60.67 while that of the cells transduced by the MS2RLP-ZsGreen1 12X particles produced solely with the p8.74AZF-MS2-Coat encapsidation plasmid is 1.18.
- the fluorescence obtained is respectively two and five times greater when the particles are produced with 50% of the p8.74AZF-MS2-Coat encapsidation plasmid and 50% of the plasmid p8.74 only when produced only with plasmid p8.74AZF-MS2-Coat.
- the p24 protein is detectable at four levels:
- the p24 protein should be detected in larger amounts in the mature state (p24) than in the precursor protein state (p160, p55) or intermediate protein precursors. Indeed, the maturation of the viral particles is done after the release of the particle by the production cell. In other words, during their production, the particles bud on the surface of the production cell, and are released from the cell in the state of immature particles. It is only after the release of the particles in the supernatant that the GAG-POL and GAG protein precursors mature into definitive proteins.
- the p24 protein is detectable at four levels:
- each precursor is heavier by 12 kDa, which corresponds to the size of the Coat protein of bacteriophage MS2 inserted into the second zinc finger of the Nucleocapsid protein.
- Figure XXXXI shows, on the ILV track, the different forms of protein precursors, p160 (GAG-POL), p55 (GAG) as well as the perfectly mature p24 protein. There is a majority of mature p24 protein, compared to protein precursor forms. On the 100/0 track, corresponding to MS2RLP-ZsGreen1 12X particles produced solely with plasmid p8.74AZF-MS2-Coat as encapsidation plasmid, the proportion of precursors / mature p24 protein increases relative to ILV , showing that the insertion of the bacteriophage MS2 Coat protein decreases the maturation of the viral particles.
- the proportion of protein precursors p172 and p67 decreases in favor of protein precursors p160 and p55, to arrive at the same level of expression for the 50/50 track.
- the addition of the plasmid p8.74 to the plasmid p8.74AZF-MS2-Coat, as encapsidation plasmid for the production of particles results in the increase of the proportion of the mature p24 protein (FIG. XXXXIb, 15 seconds exposure).
- Envelope plasmid This plasmid carries the gene encoding an envelope protein, which may be VSV-G encoding the coat protein of vesicular stomatitis virus ( Figure III).
- these plasmids are used for the production of lentiviral particles MS2 / PP7-RLP-mCherry 12X-ZsGreen1 2X.
- Plasmid expression of a sequence of interest This plasmid is identical to that described in Example 1 (whose expression cassette is as described in Figure I).
- Envelope plasmid This plasmid is identical to that described in Example 1 (whose expression cassette is as described in FIG. III).
- these plasmids are used for the production of lentiviral particles MS2RLP-mCherry 12X.
- Plasmid expression of a sequence of interest This plasmid is identical to that described in Example 7 (whose expression cassette is as described in Figure XXIII).
- Encapsidation plasmid This plasmid is identical to that described in Example 7 (whose expression cassette is as described in Figure XXXVII).
- Envelope plasmid This plasmid carries the gene encoding an envelope protein, which may be VSV-G encoding the coat protein of vesicular stomatitis virus ( Figure III).
- these plasmids are used for the production of lentiviral particles PP7RLP-ZsGreen1 2X.
- the two expression plasmids described above including the plasmid pcDNA.EF1 .mCherry.MS2 12X (whose expression cassette is illustrated in FIG. 1) and the plasmid pcDNA.EFI .ZsGreen1 .PP7 2X (whose cassette expression is illustrated in Figure XXIII) used in single or dual amounts;
- control lentiviral particles MS2- (NC) RLP 12X preferentially MS2-RLP-mCherry 12X, is carried out by transfection of the following three plasmids:
- control lentiviral particles PP7- (NC) 2X RLP preferentially PP7-RLP-ZsGreen1 2X, is carried out by transfection of the following three plasmids:
- the culture supernatant is replaced by fresh and unsupplemented DMEM medium.
- the production cells are incubated at 37 ° C / 5% C0 2 .
- the supernatant is collected four times (32h, 48h, 56h and 72h post transfection). Each recovery is clarified by centrifugation for 5 min at 3000 g before being microfiltered on a 0.45 ⁇ m filter (Stericup®, Millipore). All recoveries are then mixed to form the crude supernatant.
- the lentiviral particles are concentrated and purified according to the method P1 described in Example 1.
- the lentiviral particles are titrated as described in Example 1.
- This example is carried out using MS2 / PP7- (NC) RLP 12X 2X particles allowing the transfer of several types of RNAs and therefore the expression of several different proteins (ZsGreen1 + mCherry).
- HCT1 target cells 16 (ATCC, CCL-247) were inoculated into a 24-well plate and incubated 24h at 37 ° C / 5% CO2 and were transduced by particles MS2 / PP7- (NC) RLP 12X 2X at a dose of 10pg p24 / cell.
- the transduction by lentiviral particles is carried out in the presence of 8 ⁇ 9 ⁇ _ Polybrene®.
- An inhibitor of cellular defense mechanisms, BX795 (Invivogen) is used at a concentration of 6 ⁇ in the case of particles MS2 / PP7- (NC) RLP 12X 2X, MS2RLP-mCherry 12X and PP7RLP-ZsGreen1 2X.
- the target cells are recovered at 48 hours post-transduction and the percentage of cells expressing ZsGreen1 and mCherry is quantified by cytometry (Macs Quant VYB, Miltenyi Biotec).
- Figure XXXXI illustrates the efficacy of MS2 / PP7- (NC) RLP 12X 2X particles for the transfer of encapsidated RNA by encapsidation sequences from MS2 and PP7 bacteriophages into HCT1 target cells 16.
- the Figure shows that the proportion of bifluorescent cells is 97% after transduction of particles MS2 / PP7- (NC) RLP 12X 2X and 98% after transduction of particles MS2RLP-mCherry 12X and PP7RLP-ZsGreen1 2X at 20pg p24 / cell. The same order of transduction efficiency is therefore observed with the MS2 / PP7- (NC) RLP 12X 2X particles, for a half-dose of MS2 / PP7- (NC) RLP 12X 2X.
- the target cells transduced by the MS2RLP-mCherry 12X particles alone or PP7RLP-ZsGreen1 2X alone show a percentage of transduction efficiency similar to the percentage obtained after transduction of the target cells by the particles MS2 / PP7- (NC) RLP 12X 2X for a single fluorescent protein.
- the results thus show that the RLP particles are capable of transporting and transferring at least 2 types of packaged RNA by two different systems (PP7 and MS2) in a single target cell transduction.
- RNA The demonstration of the transfer capacity of different types of RNA directed by two different encapsidation sequences, MS2 and PP7, in a single transduction of the same batch of RLP represents a significant gain on the modulation of the types of RNA.
- Encapsidated RNA particularly for the efficient transfer of RNA coding for different antigens or for the transfer of RNA coding for antigens and for an immunomodulatory protein.
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CA3023788A CA3023788A1 (fr) | 2016-05-13 | 2017-05-12 | Particule virale pour le transfert d'arns, notamment dans les cellules impliquees dans la reponse immune |
EP17730842.6A EP3454889A2 (fr) | 2016-05-13 | 2017-05-12 | Particule virale pour le transfert d'arns, notamment dans les cellules impliquées dans la réponse immune |
CN201780043718.0A CN109562150A (zh) | 2016-05-13 | 2017-05-12 | 用于将rna转移到尤其参与免疫应答的细胞中的病毒颗粒 |
AU2017263138A AU2017263138B2 (en) | 2016-05-13 | 2017-05-12 | Viral particle for RNA transfer, especially into cells involved in immune response |
SG11201810014UA SG11201810014UA (en) | 2016-05-13 | 2017-05-12 | Viral particle for rna transfer, especially into cells involved in immune response |
US16/301,354 US11371059B2 (en) | 2016-05-13 | 2017-05-12 | Viral particle for the transfer of RNAs, especially into cells involved in immune response |
JP2018559309A JP6916209B2 (ja) | 2016-05-13 | 2017-05-12 | 特に免疫応答に関与する細胞への、rnaの転移のためのウイルス粒子 |
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WO2016049258A2 (fr) | 2014-09-25 | 2016-03-31 | The Broad Institute Inc. | Criblage fonctionnel avec systèmes crisp-cas fonctionnels optimisés |
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IL262944A (en) | 2018-12-31 |
AU2017263138B2 (en) | 2022-08-25 |
AU2017263138A1 (en) | 2019-01-03 |
CN109562150A (zh) | 2019-04-02 |
US20190203228A1 (en) | 2019-07-04 |
CA3023788A1 (fr) | 2017-11-16 |
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US11371059B2 (en) | 2022-06-28 |
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WO2017194903A3 (fr) | 2018-01-25 |
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